Effects of mitochondria and o-methoxybenzoylalanine on 3-hydroxyanthranilic acid dioxygenase activity and quinolinic acid synthesis

The use of o-methoxybenzoylalanine, a selective kynureninase inhibitor, has been proposed with the aim of reducing brain synthesis of quinolinic acid, an excitotoxic tryptophan metabolite. In liver homogenates, however, this compound caused unexpected accumulation of 3-hydroxyanthranilic acid, the product of kynureninase activity and the precursor of quinolinic acid. To explain this observation, we investigated the interaction(s) of o-methoxybenzoylalanine with 3-hydroxyanthranilic acid dioxygenase, the enzyme responsible for quinolinic acid formation. When the purified enzyme or partially purified cytosol preparations were used, o-methoxybenzoylalanine did not affect 3-hydroxyanthranilic acid dioxygenase activity. However, a significant reduction of this enzymatic activity did occur when o-methoxybenzoylalanine was tested in the presence of mitochondria. It is interesting that addition of purified mitochondria to 3-hydroxyanthranilic acid dioxygenase preparations reduced the enzymatic activity and the synthesis of quinolinic acid. In vivo, administration of o-methoxybenzoylalanine significantly reduced quinolinic acid synthesis and content in both blood and brain of mice. Our results suggest that mitochondrial protein(s) interact(s) with soluble 3-hydroxyanthranilic acid dioxygenase and cause(s) modifications in the enzyme resulting in a decrease in its activity. These modifications also allow the enzyme to interact with o-methoxybenzoylalanine, thus leading to a further reduction in quinolinic acid synthesis.     

QSAR study on inhibition of brain 3-hydroxy-anthranilic acid dioxygenase (3-HAO): A molecular connectivity approach

The ability of 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3-hydroxyanthranilic acid derivatives to reduce the production of the excitotoxin quinolinic acid (QUIN) by inhibition of brain 3-hydroxyanthranilic acid dioxygenase (3-HAO) has been investigated using molecular connectivity indices (⁰χ^v, ¹χ^v, ²χ^v). The in-vivo inhibition of 3-HAO in rat cortex (pIC₅₀, nM) is used for this purpose. The regression models obtained suggest that the degree of branching of the compounds under study have a dominant role in the observed inhibition potency. The data were used to generate quantitative structure–activity relationship (QSAR) models for estimating the potency of 3-HAO. The information obtained from the correlation should be useful in designing more potent analogues.     

Synthesis of Quinolinic Acid by 3-Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro

In mammalian peripheral organs, 3-hydroxyanthranilic acid oxygenase (3HAO), catalyzing the conversion of 3-hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HAO in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy-14C]3-hydroxyanthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chromatographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 +/- 0.5 microM for 3-hydroxyanthranilic acid and a Vmax of 73.7 +/- 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3-hydroxyanthranilic acid caused less than 25% inhibition of activity at 500 microM. Both the Fe2+ dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10-fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at -80 degrees C. Taken together, these data indicate that measurements of brain 3HAO may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.     

Effects of the 3-hydroxyanthranilic acid analogue NCR-631 on anoxia-, IL-1β- and LPS-induced hippocampal pyramidal cell lossin vitro

Summary The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the putative neuropathogen quinolinic acid (QUIN). In the present study, the neuroprotective effects of the 3-HANA analogue and 3-HAO inhibitor NCR-631 was investigated using organotypic cultures of rat hippocampus. An anoxic lesion was induced by exposing the cultures to 100% N₂ for 150 min, resulting in a pronounced loss of pyramidal neurons, as identified using NMDA-R1 receptor subunit immunohistochemistry. NCR-631 provided a concentration-dependent protective effect against the anoxia. NCR-631 was also found to counteract the loss of pyramidal neurons in two models of neuroinflammatory-related damage; incubation with either LPS (10 ng/ml) or IL-1 (10 IU/ml). The findings suggest that NCR-631 has neuroprotective properties and that it may be a useful tool to study the role of kynurenines in neurodegeneration.Abbreviations EAA excitatory amino acid - 3-HANA 3-hydroxyanthranilic acid - 3-HAO 3-hydroxyanthranilic acid 3,4-dioxygenase - IL-1 interleukin-1 - KYNA kynurenic acid - LPS lipopolysaccaride - NCR-631 4,6-dibromo-3hydroxyanthranilic acid - NMDA N-methyl-d-aspartate - QUIN quinolinic acid     

Anticonvulsant effects of the 3-hydroxyanthranilic acid dioxygenase inhibitor NCR-631

Summary. The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the pro-convulsive excitotoxin quinolinic acid. In the present study, the anticonvulsant effect of the 3-HAO inhibitor NCR-631 was investigated in models of chemically- and sound-induced seizures. Administration of NCR-631 i.c.v. at a dose of 300 nmol in Sprague-Dawley rats was found to prolong the latency of occurrence of pentylenetetrazole (PTZ)-induced seizures. Also systemic pre-treatment with NCR-631 s.c. in N.M.R.I. mice subjected to PTZ-induced seizures provided an increase in the latency until onset of seizures, concomitant with a reduction in the severity of the seizures. However, the anticonvulsant effect of NCR-631 was short lasting (15–30 min), and only observed at a dose of 250 mg/kg. A similar dose- and time-dependent anticonvulsant effect of NCR-631 was found in seizure-prone DBA/2J mice following sound-induced convulsions. Hence, the findings show that NCR-631 has anticonvulsant properties against generalized tonic-clonic seizures of different origin, suggesting that it may constitute a useful tool to study the role of kynurenines in various convulsive states. Received August 31, 1999 Accepted September 20, 1999     

4-chloro-3-hydroxyanthranilate inhibits brain 3-hydroxyanthranate oxidase

Quinolinic acid is synthesized from 3-hydroxyanthranilic acid via 3-hydroxyanthranilic acid oxidase. In liver, 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase. To determine whether 4-chloro-3-hydroxyanthranilic acid also inhibits 3-hydroxyanthranilic acid oxidase in brain, 3-hydroxyanthranilic acid was injected into the cisterna magna of rats either with or without 4-chloro-3 hydroxyanthranilic acid. 3-Hydroxyanthranilic acid increased quinolinic acid concentrations throughout the brain. 4-Chloro-3-hydroxyanthranilic acid attenuated increases in brain quinolinic acid. These observations indicate that 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase in brain.Quinolinic acid is a well established systemic metabolite of l-tryptophan which has been shown to be present in brain (Wolfensberger et al., 1983; Heyes and Markey, 1988a). QUIN has proved to be a convulsant (Lapin, 1982), neurotoxin (Schwarcz et al., 1983) and agonist of N-methyl-D-aspartate receptors (Perkins and Stone, 1983) when injected directly into the central nervous system of experimental animals. Therefore increased concentrations of QUIN in brain may have neoropathologic consequences. l-Tryptophan is converted to QUIN via the kynurenine pathway. The precursor of QUIN, 2-amino-3-carboxymuconic semialdehyde is synthesized from 3-hydroxyanthranilic acid (3-HAA) by the action of 3-hydroxyanthranilic acid oxidase (3-HAA/OX) in liver and brain (Foster et al., 1986; Okuno et al., 1987). QUIN is then formed from 2-amino-3-carboxymuconic semialdehyde by a spontaneous, non-enzymatic reaction. In liver, 3-HAA/OX is inhibited by 4-chloro-3-hydroxyantranilic acid (CL-HAA; Parli et al., 1980). In the present study, rats were given an intracisternal injection of 3-HAA and the resultant increases in regional brain QUIN concentrations quantified by gas chromatography/mass spectrometry (Heyes and Markey, 1988a,b). To determine whether CL-HAA inhibit 3-hydroxyanthranilic acid oxidase in brain, CL-HAA was co-administered with 3-HAA to see whether increases in QUIN were attenuated.     

Terminal synthesis of xanthommatin in drosophila melanogaster. IV. Enzymatic and nonenzymatic catalysis

Nonenzymatic and enzymatic catalysis of the oxidation of 3-hydroxykynurenine (and 3-hydroxyanthranilic acid) has been studied and characterized in Drosophila extracts, clearing up some of the confusion surrounding the synthesis of the brown eye pigment, xanthommatin. The genetic basis of the terminal steps in pigment synthesis remains obscure, since all mutants tested have full synthetase activity.     

Kynurenine Disposition in Blood and Brain of Mice: Effects of Selective Inhibitors of Kynurenine Hydroxylase and of Kynureninase

Abstract: To study the regulation of the synthesis of quinolinic and kynurenic acids in vivo, we evaluated (a) the metabolism of administered kynurenine by measuring the content of its main metabolites 3-hydroxykynurenine, anthranilic acid, and 3-hydroxyanthranilic acid in blood and brain of mice; (b) the effects of ( m -nitrobenzoyl)alanine, a selective inhibitor of kynurenine hydroxylase and of ( o -methoxybenzoyl)alanine, a selective inhibitor of kynureninase, on this metabolism; and (c) the effects of ( o -methoxybenzoyl)alanine on liver kynureninase and 3-hydroxykynureninase activity. The conclusions drawn from these experiments are (a) the disposition of administered kynurenine preferentially occurs through hydroxylation in brain and through hydrolysis in peripheral tissues; (b) ( m -nitrobenzoyl)alanine, the inhibitor of kynurenine hydroxylase, causes the expected changes in brain kynurenine metabolism, such as a decrease of 3-hydroxykynurenine, and an increase of kynurenic acid; and (c) ( o -methoxybenzoyl)alanine, the kynureninase inhibitor, increases brain concentration of the cytotoxic compound 3-hydroxykynurenine, and unexpectedly does not reduce brain concentration of 3-hydroxyanthranilic acid, the direct precursor of quinolinic acid. Taken together, the experiments suggest that the systemic administration of a kynurenine hydroxylase inhibitor is a rational approach to increase the brain content of kynurenate and to decrease that of cytotoxic kynurenine metabolites, such as 3-hydroxykynurenine and quinolinic acid.     

Angiotensin II Regulation of Intracellular Calcium in Astroglia Cultured from Rat Hypothalamus and Brainstem

Abstract: Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon-γ (IFN-γ) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN-γ induced the expression of indoleamine 2,3-dioxygenase (IDO) activity. Whereas tumor necrosis factor-α did not affect enzyme induction by IFN-γ, lipopolysaccharide modulated IDO activity differently in the two IFN-γ-activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3-hydroxylase activity was stimulated by IFN-γ. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN-γ markedly stimulated the activity of this enzyme only in MT2 cells. IFN-γ-treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3-hydroxyanthranilic acid quinolinic acids from l -[5-³H]tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.     

3-Hydroxyanthranilic acid metabolism. IV. Spectrophotometric evidence for the formation of an intermediate

Evidence has been presented for the formation of an intermediate compound in the metabolism of 3-hydroxyanthranilic acid to quinolinic acid by 3-hydroxyanthranilase from rat liver preparations. The production of the intermediate was demonstrated by spectrophotometric analyses and quinolinic acid measurements of incubation mixtures in which small amounts of acetone powder extracts of rat liver were used as the enzyme source. The calculated extinction coefficient of the compound was more than double that of the substrate or of the final product, quinolinic acid.The intermediate was shown to be an oxidation product of 3-hydroxyanthranilate as indicated by Thunberg experiments. The data obtained indicate that the intermediate may be a quinone-type compound.     

NIACIN BIOSYNTHESIS IN GERMINATING×LAELIOCATTLEYA ORCHID EMBRYOS AND YOUNG SEEDLINGS

The roles of tryptophan, kynurenne, 3-hydroxyanthranilic acid, and quinolinic acid as niacin precursors were investigated by assessing their germination and growth-promoting effects in orchid embryo and seedling cultures. Only niacin, 3-hydroxyanthranilic acid, quinolinic acid and kynurenine exhibited a growth-promoting effect on seedlings of all ages. Tryptophan inhibited the growth of 70-day-old seedlings but enhanced growth of older seedlings. The data presented are interpreted to be consistent with the hypothesis that orchids may synthesize their niacin by a pathway similar to that of certain bacteria, fungi, birds, and mammals.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.     

The kinetic mechanism of inhibition of liver pyridoxal kinase with tryptophan metabolites

The activity of purified liver pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) was determined in the presence of 13 different tryptophan metabolites. Only 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid and quinolinic acid were found to inhibit the enzyme with I50 values of 0.1, 0.12, 0.36 and 0.42 mM, respectively. The inhibition was not related to the presence of pyridine nucleus in the metabolites molecule as was proved from the patterns of inhibition.     

Rat 3-Hydroxyanthranilic Acid Oxygenase: Purification from the Liver and Immunocytochemical Localization in the Brain

3-Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6), the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37-38,000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical Km of 3 microM for the substrate 3-hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe2+) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double-diffusion experiments. Immunohistochemical studies using purified anti-rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brain's quinolinic acid system.     

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.     

A radioenzymatic assay for quinolinic acid

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.     

Tryptophan Loading Induces Oxidative Stress

In previous studies tryptophan loads have been administered to human subjects in order to raise central levels of 5-hydroxytryptamine (5HT) and assess the effects of 5HT on behaviour and mood. However, tryptophan is metabolised primarily along the oxidative kynurenine pathway. In this study a 6 g oral tryptophan load was administered to 15 healthy volunteers and the levels of kynurenines and lipid peroxidation products (indicative of oxidative stress) were measured. The results demonstrate that tryptophan loading produces a highly significant increase in lipid peroxidation products in parallel with increased kynurenines. The oxidative stress may result from the generation of quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are known to have the ability to generate free radicals. The results may have implications for the use of tryptophan loading in psychiatric practice, and for the chronic use of diets high in tryptophan.     

Mechanisms by which acyclovir reduces the oxidative neurotoxicity and biosynthesis of quinolinic acid

The concentration of the endogenous neurotoxin quinolinic acid (QA) is increased in the central nervous system of mice with herpes simplex encephalitis. We have previously shown that the antiherpetic agent acyclovir (AC) has the ability to reduce QA-induced neuronal damage in rat brain, by attenuating lipid peroxidation. The mechanism by which QA induces lipid peroxidation includes the enhancement of the iron (Fe)-mediated Fenton reaction and the generation of free radicals, such as the superoxide anion (O(2)(-)). Thus, the present study determined whether AC has the ability to reduce Fe(2+)-induced lipid peroxidation, O(2)(-) generation and QA-induced superoxide anion generation, and to bind free Fe. O(2)(-) and Fe(2+) are also cofactors of the enzymes, indoleamine-2,3-dioxygenase (IDO) and 3-hydroxyanthranilate-3,4-dioxygenase (3-HAO) respectively. These enzymes catalyse steps in the biosynthesis of QA; thus, the effect of AC on their activity was also investigated. AC significantly attenuates Fe(2+)-induced lipid peroxidation and O(2)(-) generation. AC reduces O(2)(-) generation in the presence of QA and strongly binds Fe(2+) and Fe(3+). It also reduces the activity of both IDO and 3-HAO, which could be attributed to the superoxide anion scavenging and iron binding properties, respectively, of this drug.     

Tryptophan loading induces oxidative stress

In previous studies tryptophan loads have been administered to human subjects in order to raise central levels of 5-hydroxytryptamine (5HT) and assess the effects of 5HT on behaviour and mood. However, tryptophan is metabolised primarily along the oxidative kynurenine pathway. In this study a 6 g oral tryptophan load was administered to 15 healthy volunteers and the levels of kynurenines and lipid peroxidation products (indicative of oxidative stress) were measured. The results demonstrate that tryptophan loading produces a highly significant increase in lipid peroxidation products in parallel with increased kynurenines. The oxidative stress may result from the generation of quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are known to have the ability to generate free radicals. The results may have implications for the use of tryptophan loading in psychiatric practice, and for the chronic use of diets high in tryptophan.